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Literature summary extracted from

  • Kaper, T.; Lebbink, J.H.G.; Pouwels, J.; Kopp, J.; Schulz, G.E.; van der Oost, J.; de Vos, W.M.
    Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus (2000), Biochemistry, 39, 4963-4970.
    View publication on PubMed

Application

EC Number Application Comment Organism
3.2.1.21 synthesis synthesis of glycoconjugates and oligosaccharides Pyrococcus furiosus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.21 expression in Escherichia coli Pyrococcus furiosus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.21 3.3 A resolution Pyrococcus furiosus
3.2.1.B28 3.3 A resolution structural model. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)8-barrel fold Pyrococcus furiosus

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.21 E417S mutant with reduced pH optimum, increased Km and turnover number for 2-nitrophenyl-beta-1,4-D-glucopyranoside Pyrococcus furiosus
3.2.1.21 F426Y mutant with unchanged pH optimum, slight increased Km and slight reduced turnover number for 2-nitrophenyl-beta-1,4-D-glucopyranoside Pyrococcus furiosus
3.2.1.21 M424K mutant with increased pH optimum, slight increased Km and reduced turnover number for 2-nitrophenyl-beta-1,4-D-galactopyranoside Pyrococcus furiosus
3.2.1.B28 E417S mutation in phosphate binding site, 5fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate. Activity on nonphosphorylated sugars is largely reduced Pyrococcus furiosus
3.2.1.B28 E417S/M424K/F426Y mutations in phosphate binding site, 3fold increase of the efficiency of hydrolyzing 2-nitrophenyl beta-D-galactopyranoside-6-phosphate. Activity on nonphosphorylated sugars is largely reduced Pyrococcus furiosus
3.2.1.B28 F426Y mutation in phosphate binding site, results in an increased affinity for galactosides. Activity on nonphosphorylated sugars is largely reduced Pyrococcus furiosus
3.2.1.B28 M424K mutation in phosphate binding site, shifting pH optimum from 5.0 to 6.0. Activity on nonphosphorylated sugars is largely reduced Pyrococcus furiosus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.21 0.28
-
 2-Nitrophenyl-beta-D-glucopyranoside
-
 Pyrococcus furiosus
3.2.1.21 2.3
-
 2-nitrophenyl-beta-D-galactopyranoside
-
 Pyrococcus furiosus
3.2.1.21 31
-
 2-nitrophenyl-beta-D-galactopyranoside-6-phosphate
-
 Pyrococcus furiosus
3.2.1.B28 0.28
-
 2-nitrophenyl beta-D-glucopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 0.51
-
 2-nitrophenyl beta-D-glucopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 0.57
-
 2-nitrophenyl beta-D-glucopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 0.89
-
 2-nitrophenyl beta-D-galactopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 2.3
-
 2-nitrophenyl beta-D-galactopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 4.8
-
 2-nitrophenyl beta-D-galactopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 16
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 22
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 29
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 31
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 38
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 40
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 42
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 59
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.21 Pyrococcus furiosus Q51723
-
-
3.2.1.B28 Pyrococcus furiosus Q51723
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.21 to homogeneity, recombinant enzyme Pyrococcus furiosus

Source Tissue

EC Number Source Tissue Comment Organism Textmining
3.2.1.21 additional information celB gene Pyrococcus furiosus
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.21 2-nitrophenyl beta-1,4-D-galactopyranoside 6-phosphate + H2O
-
 Pyrococcus furiosus ?
-
 ?
3.2.1.21 2-nitrophenyl beta-D-galactopyranoside + H2O
-
 Pyrococcus furiosus 2-nitrophenol + beta-D-galactopyranose
-
 ?
3.2.1.21 2-nitrophenyl beta-D-galactopyranoside 6-phosphate + H2O
-
 Pyrococcus furiosus 2-nitrophenol + beta-D-galactopyranose 6-phosphate
-
 ?
3.2.1.21 2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus furiosus 2-nitrophenol + D-glucopyranose
-
 ?
3.2.1.B28 2-nitrophenyl beta-D-galactopyranoside + H2O
-
 Pyrococcus furiosus 2-nitrophenol + D-galactopyranoside
-
 ?
3.2.1.B28 2-nitrophenyl beta-D-galactopyranoside-6-phosphate + H2O
-
 Pyrococcus furiosus 2-nitrophenol + D-galactopyranoside-6-phosphate
-
 ?
3.2.1.B28 2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus furiosus 2-nitrophenol + D-glucopyranoside
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.21 tetramer composed of 4 identical subunits, X-ray diffraction study Pyrococcus furiosus

Synonyms

EC Number Synonyms Comment Organism
3.2.1.21 CelB
-
 Pyrococcus furiosus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.21 49
-
 2-nitrophenyl beta-1,4-D-galactopyranoside 6-phosphate
-
 Pyrococcus furiosus
3.2.1.21 860
-
 2-Nitrophenyl-beta-D-glucopyranoside
-
 Pyrococcus furiosus
3.2.1.21 2050
-
 2-nitrophenyl-beta-D-galactopyranoside
-
 Pyrococcus furiosus
3.2.1.B28 20 50 2-nitrophenyl beta-D-galactopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 26
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 49
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 69
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 171
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 175
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 772
-
 2-nitrophenyl beta-D-glucopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 777
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 840
-
 2-nitrophenyl beta-D-glucopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 860
-
 2-nitrophenyl beta-D-glucopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 892
-
 2-nitrophenyl beta-D-galactopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1060
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1880
-
 2-nitrophenyl beta-D-galactopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 3300
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.21 4
-
 for 2-nitrophenyl-beta-1,4-D-galactopyranoside-6-phosphate Pyrococcus furiosus
3.2.1.21 5
-
 for 2-nitrophenyl-beta-1,4-D-gluco- and galactopyranoside Pyrococcus furiosus
3.2.1.B28 4
-
 wild-type, substrate 2-nitrophenyl beta-D-galactopyranoside-6-phosphate Pyrococcus furiosus
3.2.1.B28 5
-
 mutant E417S/M424K/F426Y, substrates 2-nitrophenyl beta-D-glucopyranoside, 2-nitrophenyl beta-D-galactopyranoside Pyrococcus furiosus
3.2.1.B28 5
-
 wild-type, substrates 2-nitrophenyl beta-D-glucopyranoside, 2-nitrophenyl beta-D-galactopyranoside Pyrococcus furiosus
3.2.1.B28 6
-
 mutant E417S/M424K/F426Y, substrate 2-nitrophenyl beta-D-galactopyranoside-6-phosphate Pyrococcus furiosus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.B28 0.7
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1.6
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 4.2
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 4.4
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 7.7
-
 2-nitrophenyl beta-D-galactopyranoside-6-phosphate mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 13
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S/M424K/F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 25.3
-
 2-nitrophenyl beta-D-galactopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 113
-
 2-nitrophenyl beta-D-glucopyranoside mutant E417S, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 396
-
 2-nitrophenyl beta-D-galactopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 899
-
 2-nitrophenyl beta-D-galactopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1000
-
 2-nitrophenyl beta-D-galactopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1484
-
 2-nitrophenyl beta-D-glucopyranoside mutant F426Y, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 1510
-
 2-nitrophenyl beta-D-glucopyranoside mutant M424K, pH 5.0, 90°C Pyrococcus furiosus
3.2.1.B28 4048
-
 2-nitrophenyl beta-D-glucopyranoside wild-type, pH 5.0, 90°C Pyrococcus furiosus